1. Field of the Invention
This invention relates to particles for use in assays for analytes, for therapeutic use and for use as pigments. The particles have optical and surface properties that render them more suitable for the aforementioned uses.
The clinical diagnostic field has seen a broad expansion in recent years, both as to the variety of materials of interest that may be readily and accurately determined, as well as the methods for the determination. Convenient, reliable and non-hazardous means for detecting the presence of low concentrations of materials in liquids is desired. In clinical chemistry these materials may be present in body fluids in concentrations below 10xe2x88x9212 molar. The difficulty of detecting low concentrations of these materials is enhanced by the relatively small sample sizes that can be utilized.
The need to determine many analytes in blood and other biological fluids has become increasingly apparent in many branches of medicine. In endocrinology the knowledge of plasma concentration of a number of different hormones is often required to resolve a diagnostic problem or a panel of markers for a given diagnosis where the ratios could assist in determining disease progression. An even more pressing need is evident in other areas such as allergy testing, the screening of transfused blood for viral contamination or genetic diagnosis.
In other assays such as nucleic acid hybridization assays, there is need to detect and quantify specific target and positive control sequences in a single tube without time consuming separations and transfer steps. In principle internal controls will eliminate the need for a standard curve. Amplification and detection in a single tube without opening the tube also overcomes contamination problems. In mutation analysis, the ability to measure two or more variants in a single tube would allow one to monitor quantitatively the appearance of mutant populations.
Most multi-analyte assays are heterogeneous, have poor sensitivity and poor dynamic range (2 to 100 fold difference in concentration of the analytes is determined) and some require the use of sophisticated instrumentation. A homogeneous assay that has higher sensitivity, large dynamic range (103 to 104-fold difference in analyte concentration), and fewer and more stable reagents would increase the simplicity and reliability or multianalyte assays.
Luminescent compounds, such as fluorescent compounds and chemiluminescent compounds, find wide application in the assay field because of their ability to emit light. For this reason, luminescers have been utilized as labels in assays such as nucleic acid assays and immunoassays. For example, a member of a specific binding pair is conjugated to a luminescer and various protocols are employed. The luminescer conjugate can be partitioned between a solid phase and a liquid phase in relation to the amount of analyte in a sample suspected of containing the analyte. By measuring the luminescence of either of the phases, one can relate the level of luminescence observed to a concentration of the analyte in the sample.
Particles, such as liposomes and erythrocyte ghosts, have been utilized as carriers of encapsulated water-soluble materials. For example, liposomes have been employed to encapsulate biologically active material for a variety of uses, such as drug delivery systems wherein a medicament is entrapped during liposome preparation and then administered to the patient to be treated.
Particles, such as latex beads and liposomes, have also been utilized in assays. For example, in homogeneous assays an enzyme may be entrapped in the aqueous phase of a liposome labeled with an antibody or antigen. The liposomes are caused to release the enzyme in the presence of a sample and complement. Antibody or antigen-labeled liposomes, having water soluble fluorescent or non-fluorescent dyes encapsulated within an aqueous phase vesicle or lipid soluble dyes dissolved in the lipid bilayer of a lipid, have also been utilized to assay for analytes capable of entering into an immunochemical reaction with the surface bound antibody or antigen. Detergents have been used to release the dyes from the aqueous phase of the liposomes.
Dyed latex particles have been used previously not only in immunoassays but also for other diverse uses such as photodynamic therapy and as pigments. Both absorptive dyes and dyes that impart fluorescent or chemiluminescent properties have been incorporated into these particles. Frequently, the latex is dyed by dispersing the particles in a solvent that is at least partially organic. The organic solvent causes the particles to swell and is believed to permit the incorporation of increased concentrations of the dyes. While the optical properties of the dyed particles might be expected to be enhanced as more dye is incorporated into the particles this is not necessarily the case. We have observed that increased concentrations of fluorescent dyes tend to promote self-quenching and higher concentrations of absorptive dyes can lead to ground state multimers with different optical properties from the fully dispersed state. Even at low concentrations of chemiluminescent olefins and energy acceptors, we found that the rates of decay of dioxetanes produced in latex particles upon reaction with singlet oxygen are non-linear. This observation was consistent with previously published multiphasic decay of solute excited states in polystyrene films.
2. Brief Description of the Related Art
U.S. Pat. No. 5,340,716 (Ullman, et al.) describes an assay method utilizing photoactivated chemiluminescent labels.
Photoactivatable chemiluminescent matrices are described in U.S. Pat. No. 5,709,994 (Pease, et al.).
U.S. Pat. No. 5,194,300 (Cheung 1) discusses methods of making fluorescent microspheres.
U.S. Pat. No. 5,132,242 (Cheung 2) discusses fluorescent microspheres and methods of using them.
U.S. Pat. No. 4,837,168 (de Jaeger) discloses an immunoassay using colorable latex particles.
U.S. Pat. No. 4,699,826 (Schwartz, et al.) discusses fluorescently labeled microbeads.
Madison, et al., describes latex nanosphere delivery system (LNDS), novel nanometer-sized carriers of fluorescent dyes and active agents selectively target neuronal subpopulations via uptake and retrograde transport in Brain Research (1990) 522:90-98.
U.S. Pat. No. 3,996,056 (Muller) discusses diazotype reproduction layer formed from matrix of spheric particle polystyrene pigment and diazotype components.
U.S. Pat. No. 3,755,238 (Wiita) discloses high gloss and low block coating composition containing plasticized vinyl resin latex and finely divided polyolefin particles.
O""Connell, et al., describe a highly sensitive immunoassay system involving antibody-coated tubes and liposome-entrapped dye in Clin. Chem. (1 985) 31(9):1424-1426.
U.S. Pat. No. 5,618,732 (Pease, et al. 1) discusses a method of calibration with photoactivatable chemiluminescent matrices.
U.S. Pat. No. 5,489,537 (Van Aken) discloses agglutination assays and kits employing colloidal dyes.
U.S. Pat. No. 5,284,752 (Sutton 1) discusses methods of preparing a polymeric latex composition and water-insoluble biological reagent.
U.S. Pat. No. 5,234,841 (Sutton 2) discloses methods of preparing a polymeric latex composition and water-insoluble biological reagent.
U.S. Pat. No. 5,157,084 (Lee, et al.) discusses a process of making hollow polymer latex particles.
U.S. Pat. No. 5,053,443 (Sutton 3) discloses methods of preparing a polymeric latex composition and water-insoluble biological reagent.
U.S. Pat. No. 4,891,324 (Pease) discloses a particle with luminescer for assays.
U.S. Pat. No. 4,801,504 (Burdick, et al.) discusses fluorescent labels having a polysaccharide bound to polymeric particles.
U.S. Pat. No. 4,784,912 (Schaeffer, et al.) discusses latex particles incorporating stabilized fluorescent rare earth labels.
U.S. Pat. No. 4,650,770 (Liu, et al.) discloses energy absorbing particle quenching in light emitting competitive protein binding assays.
U.S. Pat. No. 4,483,929 (Szoka) discusses liposomes with glycolipid-linked antibodies.
U.S. Pat. No. 4,388,296 (Hart) discloses energy-emifting latex particulates.
U.S. Pat. No. 4,318,707 (Litman, et al.) discusses macromolecular fluorescent quencher particle in specific receptor assays.
U.S. Pat. No. 4,264,766 (Fischer) discloses immunological diagnostic reagents.
Latex particles in analytical reagents, elements and methods is discussed in European Patent Application 0 360 452 A2 (Warshawsky, et al.).
Aqueous suspension for diagnostic tests is discussed in European Patent Application 0 369 515 A1 (Brouwer).
U.S. Pat. No. 5,573,909 (Singer, et al.) discloses fluorescent labeling using microparticles with controllable Stokes shift.
Hydrophilic latex particles and use thereof is discussed by Batz, et al., in U.S. Pat. No. 4,415,700.
Colored latex and methods for making the same and colored finely divided products are discussed in U.S. Pat. No. 4,487,855 (Shih, et al.).
U.S. Pat. No. 4,419,453 (Dorman, et al.) discloses immunological agglutination assays with dyed or colored latex and kits.
U.S. Pat. No. 4,745,075 (Hadfield, et al.) discusses diagnostic test methods.
A process for producing immunological diagnostic reagents is discussed in U.S. Pat. No. 4,181,636 (Fischer).
U.S. Pat. No. 5,716,855 (Lerner, et al.) discloses fluorescent latex containing at least two fluorochromes, process for producing it and application thereof.
One aspect of the present invention is a light emitting composition comprising a polymeric matrix having dissolved therein a photoactive compound. The composition has the characteristic that, after activation of the photoactive compound, the rate of decrease in the intensity of light emission from the composition at any time during a 20-fold decrease in the intensity is proportional to the intensity of the light emission. In one embodiment the polymeric matrix is comprised of particles of about 20 nm to about 100 xcexcm in diameter.
Another aspect of the present invention is a light emitting composition comprising particles of about 20 to about 100 xcexcm in diameter. The particles comprise a polymeric matrix having dissolved therein (a) about 1 to about 20% by weight of a dopant and (b) a photoactive compound. The light emitting composition is characterized in that, following cessation of activation of the photoactive compound, the rate of decrease in the intensity of light emission from the polymeric matrix at any time during a 20-fold decrease in the intensity is proportional to the intensity of the light emission.
Another aspect of the present invention is a method for determining an analyte. A combination is provided comprising (1) a medium suspected of containing the analyte, (2) a first specific binding pair (sbp) member capable of binding to the analyte or to a second sbp member to form a complex related to the presence of the analyte, wherein at least one of said sbp members is bound to polymeric particles of about 20 nm to about 100 xcexcm in diameter having homogeneously dispersed therein about 1 to about 20 weight percent of a dopant and a photoactive substance. The photoactive substance is activated and the effect of the activating on the optical properties of the combination is determined. The magnitude of the effect is related to the amount of the analyte in the medium.
Also included with the scope of the present invention is a kit for use in an assay. The kit comprises in packaged combination reagents for conducting an assay. The reagents comprise at least one member of a specific binding pair bound to polymeric particles of about 20 nm to about 100 xcexcm in diameter having incorporated therein about 1 to about 20 weight percent of a dopant and a photoactive substance.